Molecular interactions between the surfaces of lymphocytes and endothelial cells play a critical role in the extravasation (migration into tissue) of lymphocytes from the blood stream (Springer, 1990, Nature 346:425; Pober and Cotran, 1990, Transplantation 50:537). Studies from this (Dustin and Springer, 1988, J. Cell Biol. 107:321; de Fougerolles et al., 1991, J. Exp. Med. 174:253) and other laboratories (Elices et al., 1990, Cell 60:577; Schwartz et al., 1990, J. Clin. Invest. 85:2019) demonstrate that two members of the integrin family of cell-surface heterodimers--lymphocyte function-associated antigen-1 (LFA-1) and very late activation antigen-4 (VLA-4)--mediate distinct mechanisms for lymphocyte-endothelial cell adhesion. LFA-1, whose expression is limited to leukocytes, can bind to intercellular adhesion molecule-1 (ICAM-1) or to ICAM-2 on the surface of stimulated or unstimulated endothelial cells (Springer, 1990, Nature 346:425). ICAM-1 expression by endothelial cells in culture is substantially upregulated following stimulation by pro-inflammatory cytokines such as TNF, IL-1, or INF-.gamma. (Dustin and Springer, 1988, J. Cell Biol. 107:321; Pober et al., 1986, J. Immunol. 137:1893). ICAM-2 expression is constitutively high in vitro and not upregulated by cytokines (de Fougerolles et al., 1991, J. Exp. Med. 174:253; Staunton et al., 1989., Nature 339:61). ICAM-1 and ICAM-2 are also constitutively expressed by peripheral blood lymphocytes, with ICAM-1 showing a significant increase in expression following cell activation (de Fougerolles et al., 1991, J. Exp. Med. 174:253). Interactions between LFA-1 and its counter-receptors have been implicated in a number of lymphocyte functions, including cytotoxic T lymphocyte killing, delivery of T cell help, B lymphocyte responses, and graft rejection, as well as the adherence of lymphocytes and neutrophils to endothelial cells, fibroblasts, or epithelial cells (Springer, 1990, Nature 346:425; Kishimoto et al., 1989, Adv. Immunol. 46:149; Springer et al., 1987, Annu. Rev. Immunol. 5:223; Carlos and Harlan, 1990, Immunol. Rev. 114:1).
The integrin VLA-4, that contains the .alpha.4 (CD49d) subunit noncovalently associated with the .beta.1 (CD29) subunit, is expressed by lymphocytes, monocytes, and neural crest-derived cells, and can interact with vascular cell adhesion molecule-1 (VCAM-1) (Elices et al., 1990, Cell 60:577). Like ICAM-1 and ICAM-2, VCAM-1 is a member of the immunoglobulin (Ig) superfamily (Osborn et al., 1989, Cell 59:1203), but unlike the ICAMs, VCAM-1 is not expressed by lymphocytes (Wellicome et al., 1990, J. Immunol. 144:2558; Rice et al., 1990, J. Exp. Med. 171:1369). VCAM-1 expression is very low or absent on resting endothelial cells in culture but can be induced by cytokines such as TNF or IL-1 with kinetics of induction similar but not identical to that of ICAM-1 (Wellicome et al., 1990, J. Immunol. 144:2558; Carlos et al., 1990, Blood 76:965). Peak expression of VCAM-1 after continuous treatment of endothelial cells with TNF in culture occurs somewhat earlier than the peak expression of ICAM-1, but both persist at levels substantially higher than basal expression for at least 48 hr (Carlos et al., 1990, Blood 76:965). Unlike LFA-1, however, VLA-4 can also interact with fibronectin, binding to the alternatively spliced CS-1 region located C-terminal to the RGD site of fibronectin recognized by the integrin VLA-5 (Guan and Hynes, 1990, Cell 60:53; Wayner et al., 1989, J. Cell Biol. 109:1321; Hemler, 1990, Annu. Rev. Immunol. 8:365). VLA-4 and its counter-receptors have been implicated in a number of physiologic and pathophysiologic processes in addition to lymphocyte-endothelial cell adhesion including cytotoxic T cell killing (Clayberger et al., 1987, J. Immunol. 138:1510), lymphopoiesis (Miyake et al., 1991, J. Exp. Med. 173:599; Williams et al., 1991, Nature 352:438; Miyake et al., 1991, J. Cell Biol. 114:557; Ryan et al., 1991, J. Clin. Invest. 88:995), germinal center development (Freedman et al., 1990, Science 249:1030), tumor metastasis (Taichman et al., 1991, Cell Regul. 2:347; Rice and Bevilacqua, 1989, Science 246:1303), atherogenesis (Cybulsky and Gimbrone, 1991, Science 251:788), and acute graft rejection (Briscoe et al., 1991, Transplantation 51:537).
Recent studies have demonstrated that two different VCAM-1 precursors can be produced by alternative mRNA splicing (Cybulsky et al., 1991, Proc. Natl. Acad. Sci. U.S.A. 88:7859; Polte et al., 1990, Nucleic Acids Res. 18:5901; Cybulsky et al., 1991, Am. J. Pathol. 138:815; Hession et al., 1991, J. Biol. Chem. 266:6682). The original VCAM-1 cDNA clone encodes a transmembrane glycoprotein with six predicted immunoglobulin-like domains (VCAM-6D) (Osborn et al., 1989, Cell 59:1203). Several subsequently identified VCAM-1 cDNA clones, which were produced from stimulated HUVEC using polymerase chain reaction, differ from the original clone by containing a 276 base-pair insert predicted to encode an additional immunoglobulin-like domain after the first three domains of VCAM-1, suggesting a seven-domain form of VCAM-1 (VCAM-7D) (Polte et al., 1990, Nucl. Acids Res. 18:5901; Cybulsky et al., 1991, Am. J. Pathol. 138:815; Hession et al., 1991, J. Biol. Chem. 266:6682). The two forms of VCAM-1 MRNA most likely represent alternatively spliced products of the same precursor MRNA.
There is increasing evidence for multiple ligand recognition by integrins. A strategy comparing the inhibitory effects of receptor and counter-receptor monoclonal antibody (mAb) was used to provide evidence for LFA-1 counter-receptors distinct from ICAM-1 (Dustin et al., 1988, J. Cell. Biol. 107-321; Rothlein et al., 1986, J. Immunol. 137:1270; Makgoba et al., 1988, Eur. J. Immunol. 18:637), and led to the subsequent identification of ICAM-2 and ICAM-3 (de Fougerolles and Springer, 1991, J. Exp. Med. 174:253; Staunton et al., 1989, Nature 339:61; de Fougerolles and Springer, 1991, J. Exp. Med. In press). Although VLA-4 has been shown to bind to fibronectin and VCAM-1, it has not been known whether VLA-4 interacts with other ligand(s) completely distinct from VCAM-1.
Adhesion to endothelium that is dependent on .alpha.4 integrin(s) and that is not ascribable to VCAM-1 or fibronectin has not been suggested by the prior art. One previous study of resting T cell adhesion to stimulated HUVEC found no difference in inhibition between the anti-VCAM-1 mAb 4.beta.9 and a function-blocking anti-VLA-4 mAb (Oppenheimer-Marks et al., 1991, J. Immunol. 147:2913). In a study (Schwartz et al., 1990, J. Clin. Invest. 85:2019) of LFA-1-negative B cells obtained from a patient with leukocyte adhesion deficiency (Anderson and Springer, 1987, Annu. Rev. Med. 38:175), mAb 4B9 failed to inhibit binding to stimulated HUVEC as well as a function-blocking anti-VLA-4 mAb; the difference was attributed to lymphocyte interactions with fibronectin on HUVEC. In studies (Graber et al., 1990, J. Immunol. 145:819; Thornhill et al., 1991, J. Immunol. 146:592) of other anti-VCAM-1 mAb used with various mAbs that block LFA-1 function, binding of resting T cells to stimulated HUVEC was found to be inhibited but not to the same level as binding to unstimulated HUVEC in the presence of the same mAbs. In one study of a lymphocytic cell line (Pulido et al., 1991, J. Biol Chem. 266:10241), mAb 4B9 and HP2/1 inhibited binding to stimulated HUVEC equally well. In another study of cell lines (Carlos et al., 1990, Blood 76:965), however, binding to stimulated HUVEC after preincubation with 4B9 and a mAb to LFA-1 .beta. chain was greater than binding to unstimulated HUVEC after preincubation with the same mAb.
Citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.